tbuckley/cabot-search huggingface dataset:
CSIG-30. EPIGENETIC REACTIVATION OF BAI1 BLOCKS ONCOGENIC IGF1R SIGNALLING IN MEDULLOBLASTOMA
Authors: Takahiro Yamamoto, Satoru Osuka, Erwin Van Meir
Journal: Neuro-Oncology (2022)

Abstract Medulloblastoma (MB) is the most aggressive pediatric brain tumor and a better understanding of this disease is warranted to develop new therapeutic approaches. Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is an orphan seven-transmembrane G protein-coupled receptor predominantly expressed in the brain and epigenetically silenced in MB formation. MDM2 is an E3 ubiquitin ligase that leads to the proteosomal degradation of multiple proteins. We previously found that BAI1 can prevent MDM2 nuclear activity by trapping it at the cell surface, and in the process suppress tumor formation by stabilizing p53. Since MDM2 also ubiquitinates cell surface proteins, we hypothesized that its membrane trapping might increase the degradation of oncogenic tyrosine kinase receptors. To explore this idea, we examined IGF1R, a known cell surface MDM2 target and found that BAI1 negatively regulates IGF1R at the protein level. In contrast, BAI1 expression did not change the protein expression levels of other RTKs (INSR, EGFR). Using co-immunoprecipitation assays we confirmed that MDM2 interacts with both BAI1 and IGF1R in MB cells. In addition, the interaction between IGF1R and β-Arrestin, which is crucial for ubiquitination and down-regulation of IGF1R by acting as an adaptor for MDM2, was increased with BAI1 expression. To examine the impact of BAI1 overexpression on IGF1 receptor signaling, we examined its downstream signaling mediators (Stat3, Akt, Erk). Stat3 and Akt phosphorylation were suppressed upon BAI1 expression, while Erk signaling was activated but through a mechanism independent from IGF1R expression. As IGF1R is known to drive radiation resistance, we examined whether BAI1 could radiosensitize the cells and found that this was indeed the case. Altogether, our data indicate that reactivating BAI1 in MB has dual anti-tumor effects by stabilizing p53 and blocking IGF1R signalling.
BIOM-48. HOXD12 IS AN INDEPENDENTLY PROGNOSTIC AGE-ASSOCIATED INDICATOR FOR POOR SURVIVAL IN OLIGODENDROGLIOMA
Authors: Nicholas Nuechterlein, Sadie Cimino, Sonali Arora, Linda Shapiro, Eric Holland, Mark Gilbert, Patrick Cimino
Journal: Neuro-Oncology (2022)

Abstract BACKGROUND Oligodendroglioma, IDH-mutant and 1p/19q-codeleted has variable outcomes that are strongly influenced by patient age. This study aimed to discover novel molecular biomarkers that may characterize an aggressive subgroup of oligodendroglioma that is associated with, but independently prognostic of patient age. METHODS Differential gene expression analysis, gene ontology analysis, and gene set enrichment analysis were conducted on TCGA RNA-seq data (N=169) to identify genes and pathways that differ significantly between older and younger patients. Genes whose expression was associated with age and survival in multivariate analyses were further studied using DNA methylation (N=171). The prognostic value of their expression and methylation profiles was then compared to known genomic biomarkers, radiographic features, and histopathologic features. RNA-seq and DNA methylation validation data were obtained from the CGGA (N=44) and a cohort published by Capper et al. (N=144), respectively. RESULTS Highly activated pathways in older oligodendrogliomas were linked to developmental transcription factors. Overexpression of HOXD12 was associated with patient age and survival in the TCGA (FDR&amp;lt; 0.01, FDR=1e-5) and the CGGA (p=0.03, p&amp;lt; 1e-3). Hypermethylation of HOXD12 was associated with age, tumor grade, and survival in the TCGA (p&amp;lt; 1e-5, p&amp;lt; 0.001, p&amp;lt; 1e-3) and with age and tumor grade in Capper et al. (p&amp;lt; 0.02, p=0.001). In the TCGA, HOXD12 hypermethylation and overexpression were independently prognostic of NOTCH1 and PIK3CA mutations, loss of 15q, and MYC activation as well as mitotic figures, Ki-67 index, microvascular proliferation, and necrosis. HOXD12 overexpression was also independently prognostic of T1-post contrast enhancement, whole tumor volume, and peritumoral edema volume. We developed a risk-stratification nomogram predicting 5- and 10-year overall survival using patient age, HOXD12 methylation, and WHO grade. CONCLUSIONS HOXD12 overexpression and hypermethylation are associated with an aggressive subtype of oligodendroglioma and may serve as superior prognostic biomarkers than previously reported genomic, radiographic, and histopathologic features.
NCOG-11. EXTENT OF RESECTION OF CONTRAST-ENHANCING AND NON-ENHANCING TUMOR: DIFFERENT IMPACTS ON SURVIVAL ACCORDING TO TYPES OF ADULT-TYPE DIFFUSE GLIOMAS IN 2021 WHO CLASSIFICATION
Authors: Yae Won Park, Sung Soo Ahn, Eui Hyun Kim, Jinna Kim, Seok-Gu Kang, Jong Hee Chang, Se Hoon Kim, Seung-Koo Lee
Journal: Neuro-Oncology (2022)

Abstract BACKGROUND To evaluate the impact of extent of resection (EOR) of contrast-enhancing (CE) and nonenhancing (NE) tumors in the types of adult-type diffuse gliomas according to the 2021 WHO classification. METHODS Total 1,196 adult-type diffuse glioma patients diagnosed between 2001 and 2021 from a single institution were enrolled (183 patients with isocitrate dehydrogenase [IDH]-mutant and 1p/19q codeleted oligodendroglioma [herein oligodendroglioma], 211 patients with IDH-mutant astrocytoma, and 802 patients with IDH-wildtype glioblastoma). Analyses were performed within each type to assess predictors of overall survival, including clinical, imaging data, histological grade, O6-methylguanine-methyltransferase (MGMT) promoter methylation status, adjuvant treatment, and EOR of CE and NE tumors. Subgroup analyses were performed in patients with CE tumor. RESULTS Among 1,196 patients, 926 (77.4%) patients had CE tumors. In oligodendrogliomas, gross total removal (GTR) of CE tumor was associated with survival (hazard ratio [HR]=0.09, P&amp;lt; 0.001), whereas GTR of NE tumor was not associated with survival (HR=0.41, P=0.088). In 81 (43.3%) oligodendroglioma patients with CE tumor, GTR of CE tumor was the only independent predictor of survival (HR=0.20, P=0.092) in multivariable analysis. GTR of CE and NE tumors was independently associated with better survival in IDH-mutant astrocytoma and IDH-wildtype glioblastoma (all Ps&amp;lt; 0.05). CONCLUSIONS GTR of both CE and NE tumors may significantly improve survival within all types of adult-type diffuse gliomas except oligodendrogliomas. In oligodendrogliomas, the EOR of CE tumor may be crucial in survival; aggressive GTR of NE tumor may be unnecessary, whereas GTR of the CE tumor is recommended.
EXTH-78. CRISPR/CAS9 KNOCKOUT SCREENS UNRAVEL EPIGENETIC REGULATORS AS DRUGGABLE DEPENDENCIES FOR SONIC HEDGEHOG MEDULLOBLASTOMA
Authors: Foteini Tsiami, Federica Piccioni, David Root, Pratiti Bandopadhayhay, Rosalind Segal, Ghazaleh Tabatabai, Daniel Merk
Journal: Neuro-Oncology (2022)

Abstract Sonic hedgehog medulloblastoma (SHH-MB) is a malignant, highly heterogeneous brain tumor entity, accounting for 30% of all MBs in the pediatric population and is characterized by aberrant activation of the canonical SHH signaling pathway. Although current therapies targeting Smoothened (Smo) have proven a promising treatment approach for SHH-MB patients, pre-existing or acquired resistance impedes its clinical efficacy. Therefore, novel targeted approaches that overcome mechanisms of resistance are urgently needed. Here, we performed a genome-wide CRISPR/Cas9 knockout screen in a murine and a human SHH-MB cell line, SMB21 and DAOY, respectively, in order to decipher tumor-specific genetic essentialities. Our data demonstrate that SMB21 cells highly depend on key mediators of the SHH pathway, such as Gli2) for their proliferation, as opposed to DAOY cells, suggesting that the latter does not represent a faithful model of SHH-MB. Among other dependencies for SHH-MB are members of the epigenetic machinery such as Dnmt1) and Smarca5). Pharmacologically, we show that DNMT1 inhibition is efficacious at clinically relevant concentrations against Smo inhibitor- sensitive, as well as resistant SHH-MB cell lines. By performing RNA sequencing of SMB21 cells, we identified early and late alterations in global gene expression induced by DNMT1 inhibition, including decreased expression of positive regulators of SHH signaling. An additional knockout drug screen in SMB21 cells unraveled synthetic lethal interactors for DNMT1 inhibitors, as validated in vitro) by drug combination treatments. Further global DNA methylation profiling in SMB cells will help to define the molecular basis of sensitivity to DNMT1 inhibitors in SHH-MB. Last but not least, genetic ablation of epigenetic regulators and combinatorial treatments using DNMT1 inhibition will be investigated in in vivo) established mouse models of SHH-MB. Summarizing, our data indicate the potential of inhibiting epigenetic regulators as novel therapeutic avenues in SHH-MB sensitive, as well as resistant to Smo inhibition.
TMET-10. FATTY ACID METABOLISM REGULATES IMMUNE REACTIVITY OF IRRADIATED GLIOBLASTOMA
Authors: Mara De Martino, Camille Daviaud, Evagelia Laiakis, Claire Vanpouille-Box
Journal: Neuro-Oncology (2022)

Abstract Radiation therapy (RT) is the standard-of-care for glioblastoma (GBM) and is the only treatment for inoperable brain tumors. In multiple cancer, RT stimulates anti-tumor immunity by at least activating cancer cell-intrinsic type I interferon (IFN-I) responses. However, GBM inevitably recur, suggesting that RT promotes immune evasion mechanisms. Altered fatty acid (FA) metabolism is an emerging mechanism that can account for treatment resistance and immune escape of GBM. Therefore, we hypothesize that RT induces a metabolic shift toward the production of FAs to foster immunosuppression of GBM. Supporting this hypothesis, our data show that RT upregulates the expression of the fatty acid synthase (FASN) and the activity of the fatty acid desaturase 2 (FADS2) to generate FAs in murine GBM models, GL261 and CT2A. To determine whether FA metabolism was preventing RT-induced IFN-I, we impaired FA synthesis by inhibiting either FASN or FADS2 using genetic (CRISPR-Cas9) or pharmacological inhibitors (FASNi or FADS2i). As expected, protein quantification (ELISA) as well as gene expression (RT-qPCR) revealed that irradiated GBM cells released greater levels of IFN-beta 24hrs post RT; an effect that was more pronounced when FASN or FADS2 were blocked. Reinforcing the role of FA metabolism in regulating immune activation, in situ immunofluorescence of irradiated intracranial GL261 tumors showed that mice treated with FASNi improved the recruitment of CD8+ T cells into GBM. Along similar lines, flow cytometry of digested GL261 tumors revealed an increase infiltration of CD11c+ CD103+ dendritic cells as well as activated CD86+ CD11b+ cells when FAs synthesis was blocked with FADS2i. Altogether, our data suggest that RT-induced FA synthesis represents a mechanism of resistance by preventing IFN-I and promoting immunological evasion of GBM. Targeting FA metabolism is a promising strategy to promote anti-tumor immunity and sensitize irradiated GBM to immunotherapies.
IMMU-10. EXPLORING THE IMMUNOLOGIC CONSEQUENCES OF ATRX DEFICIENCY IN GLIOMA
Authors: Benjamin Whitfield, Jason Huse, David Irvin, Sharvari Dharmaiah, Prit Benny Malgulwar, Hannah Roberts
Journal: Neuro-Oncology (2022)

Abstract ATRX is a key chromatin regulator, which is mutated in large subsets of both adult and pediatric gliomas. Despite being a common mutation, little is known about the biological ramifications of ATRX deficiency. Recently, it has been demonstrated that ATRX deficiency drives increased replication stress, DNA damage, and global epigenetic dysregulation. Despite these advances, little is known about the impact of ATRX deficiency on the tumor microenvironment (TME). In order to explore the impact of ATRX deficiency on the TME we utilized the RCAS/nTVa system to generate a novel murine model of ATRX deficient glioma. Mice bearing allografts of these tumors displayed significantly increased survival relative to the ATRX intact lines. This survival benefit persisted in Nu/nu mice, which lack an adaptive immune system, but not in SCID mice, which lack both adaptive and innate immunity. Bulk RNA sequencing revealed the ATRX deficient tumors displayed increased expression of inflammatory and innate immune gene sets, and western blotting revealed increased phosphorylation of tank binding kinase 1 (TBK1), a key innate immune regulator. Multiplex cytokine analysis of conditioned media also revealed increased expression of inflammatory cytokines such as CCL2, CCL5, and CXCL10 in the supernatant of ATRX deficient cell lines. Taken together, these results indicate that ATRX deficiency can drive innate immune activation, and that this activation can increase survival in vivo. Further exploration is needed to characterize the upstream drivers of TBK1 activation and elucidate alterations to immune cell infiltration in ATRXdeficient tumors.
MODL-42. ESTABLISHMENT AND CHARACTERIZATION OF A NOVEL MOUSE SYNGENEIC GLIOBLASTOMA CELL LINE
Authors: Juan Ye, Herui Wang, Chunzhang Yang, Zhengping Zhuang
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma (GBM) is the most common and the deadliest type of brain tumor affecting the adult central nervous system, carrying a median survival of 14 months. In vivo syngeneic animal models of GBM that recapitulate human GBM are urgently needed to decipher glioma biology and develop novel therapeutics, including immunotherapy. Here, we generated a syngeneic mouse glioblastoma stem cell line (RCAS-TVA-GSC01) through repeated in-vitro passages of stem-like tumor cells isolated from the RCAS-TVA glioma mouse model, which overexpresses PDGFA and carries a homozygous deletion of Ink4a-Arf locus. We characterized this new GSC line both in vitro and in vivo. This cell line expresses stemness markers CD133 and Nestin, and is able to differentiate into astrocyte, neuron, and oligodendrocyte-like cells in 2% FBS differentiating medium with markers GFAP, β-III tubulin, and O4, respectively. Transcriptomic and genomic profiling of RCAS-TVA-GSC01 cells revealed molecular heterogeneity comparable to human glioblastoma. When intracranially transplanted into wild-type C57BL/6 mice, this cell line formed high-grade gliomas with a median survival time of 56.5 days. The differentiation potential and molecular heterogeneity make RCAS-TVA GSC01 line a unique and useful tool for studying multiple aspects of glioblastoma pathogenesis and a promising platform for testing new GBM therapeutics study, especially for immunotherapy that requires a syngeneic mouse model.
NIMG-18. A GROUND TRUTH COMPARISON OF PATHOLOGICALLY CONFIRMED GLIOBLASTOMA MARGINS TO CONTRAST ENHANCEMENT AT AUTOPSY
Authors: Allison Lowman, Samuel Bobholz, Michael Brehler, Savannah Duenweg, Fitzgerald Kyereme, Elizabeth Cochran, Dylan Coss, Jennifer Connelly, Wade Mueller, Mohit Agarwal, Anjishnu Banerjee, Peter LaViolette
Journal: Neuro-Oncology (2022)

Abstract PURPOSE Glioblastoma is one of the most common and deadly adult brain tumors. Current standard treatment is surgical resection followed by radiation and concomitant chemotherapy (chemoRT). Glioblastoma progression is monitored using MRI, primarily relying on post-contrast T1-weighted imaging (T1C). Unfortunately, tumor invasion is known to extend beyond traditional contrast enhancement. T1-subtraction (T1S) maps have been introduced as a better tumor volume estimate. In this study we compare T1S map derived tumor annotations to a radiologist for identifying histologically confirmed tumor in patients with differing treatment histories at autopsy. METHODS Ten patients with autopsy confirmed glioblastoma and MRI within 1 month of death were recruited for this study. Seven patients received chemoRT combined (chemoRT+) and three patients received no treatment beyond surgery (chemoRT-). Patient’s brains were sliced axially in the same orientation as their final MRI using a patient-specific slicing jig. Large tissue samples were taken, processed, embedded in paraffin, stained for hematoxylin and eosin, and digitized at 40x resolution. Digital images were annotated for infiltrative tumor, pseudopalisading necrosis, and necrosis without palisading cells. T1S and radiologist annotations were created for each patient using their final MRI (mean 18 days prior to death). The annotated histology images were aligned and resampled into MRI space using custom software and the overlap of pathologically confirmed tumor and MRI derived annotations was compared. RESULTS T1S maps alone were significantly better at identifying areas of histologically confirmed tumor in chemoRT+ patients compared to chemoRT- patients (p=0.043). T1S derived annotations overlapped with 52% of histologically confirmed tumor in the chemoRT- patients and 78% in the chemoRT+. The radiologist drawn tumor masks were more accurate in chemoRT+ patients, identifying 61% confirmed tumor (trending, p=0.097, chemoRT+=61%). CONCLUSION These results demonstrate the difficulty of identifying pathologically confirmed tumor outside contrast enhancement in glioblastoma patients, even in the untreated state.
EPCO-24. MULTI-OMIC PROFILING OF PATIENT-DERIVED SUBCLONES IDENTIFIES AGGRESSIVE CELLULAR SUBPOPULATIONS IN PAEDIATRIC DIFFUSE HIGH GRADE GLIOMAS (PDHGGS)
Authors: Ketty Kessler, Yura Grabovska, Anna Burford, Sara Temelso, Haider Tari, Valeria Molinari, Shauna Crampsie, Lu Yu, Jyoti Choudhary, Paula Proszek, Mike Hubank, Alan Mackay, Chris Jones
Journal: Neuro-Oncology (2022)

Abstract Paediatric-type diffuse high-grade gliomas are classified into distinct subgroups based upon their location and defining molecular alterations, with very poor clinical outcomes in patients &amp;gt;3yrs. This extensive inter-tumour heterogeneity is further complicated by a wide diversity of genotypically- and phenotypically-distinct subclonal populations within individual tumours, providing a substantial barrier to developing effective treatments. We aimed to understand the dynamic cellular make-up of PDHGGs such that novel strategies aimed at targeting specific subpopulations based upon their contribution to disease progression as whole may be employed. Two complementary approaches have been undertaken to address this – first by carrying out single-cell profiling of bulk specimens, and the second isolation and propagation of single-cell-derived stem cell-like cultures in vitro. To-date we have studied 10 cases and a total of 218 subclonal colonies from both DMG-H3K27 and DHG-WT. In a spinal metastatic case of DMG-H3K27, lpWGS/FISH highlighted subpopulations driven by mis-segregation of amplified oncogenic ecDNA, and mutually exclusive subpopulations defined by MYCN, PDGFRA and CCND1. Through integrated analysis of scRNA-seq and scATAC-seq, we show distinct chromatin accessibility profiles to underlie gene expression signatures defining unique subpopulations of cells. In addition to cycling populations and those associated with lineage-specificity, we identified ‘aggressive’ subpopulations defined by significant upregulation of immediate early response genes such as FOS/FOSL1/JUN, those associated with promotion of invasion/migration such as SERPINs and MMPs. These subpopulations could be mapped to isolated single-cell-derived subclones with highly proliferative and/or motile phenotypes, defined by comprehensive profiling of expressed and secreted proteins. Differential cis-regulation driving cell identity/tumorigenesis was found in one example to occur via a trans-histone mechanism mediated by an H4-lysine-methyltransferase, KMT5B. Application of functionally-defined interventional strategies aimed at disrupting the interactions between these subpopulations based upon evolutionary biology principles may offer a novel approach to treat these otherwise incurable tumours in children and young adults.
DDDR-21. REGULATORY REDUCTASE SUBUNIT RRM2 AND DEHYDROGENASE IMPDH2 INTERACT IN GLIOBLASTOMA IN A TMZ-DEPENDENT MANNER
Authors: Graysen McManus, Ella Perrault, Jack Shireman, Divya Ventarapragada, Linna Lahmadi, Peiyu Lin, Eunis Ali, Cheol Park, Shivani Baisiwala, David James, Issam Ben-Sahra, Sebastian Pott, Anindita Basu, Jason Miska, Atique Ahmed
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma (GBM) is an aggressive primary brain tumor characterized by poor prognostic outcomes. Nearly all GBM tumors recur, due to cellular resistance against chemotherapies, including temozolomide (TMZ), which develops during active treatment. To investigate these adaptive mechanisms, our lab conducted a single-cell RNA sequencing analysis utilizing an in vivo patient-derived xenograft (PDX) model of GBM, prior to, during, and post TMZ-based therapy. Our data revealed 149 genes to be uniquely expressed in the during-TMZ condition (p &amp;lt; 0.0001), including two regulatory enzymes involved in purine metabolism: the Ribonucleotide Reductase (RNR) Regulatory Subunit 2, RRM2, and inosine monophosphate dehydrogenase 2, IMPDH2. Previous work in this lab has independently established both RRM2 and IMPDH2 as significant drivers of TMZ-resistance in GBM, through their respective abilities to alter tumor metabolism during therapy. Network analysis of genetic pathways enriched during TMZ therapy revealed a previously unidentified interaction between IMPDH2 and the RRM1 subunit of the RNR enzyme. Given these data, we aimed to determine if there was also an interaction between IMPDH2 and RRM2 in GBM. Immunocyto- and histochemistry of GBM-PDX primarily demonstrated significant co-localization between RRM2 and IMPDH2 during TMZ therapy. Immunoprecipitation (IP) and reverse-IP analyses were subsequently conducted, and revealed a previously unreported molecular interaction between the two proteins in GBM lines, which increased in a TMZ-dependent manner. These results were not corroborated in neural stem cell lines. Additionally, immunoblot analyses revealed that in RRM2-knockdown GBM-PDX, IMPDH2 protein expression is decreased compared to controls. We hypothesize the dynamic interaction of RRM2 and IMPDH2 to enhance the metabolic adaptations underlying GBM chemoresistance. The efforts of this study are now focused on investigating the potentially synergistic mechanisms of FDA-approved RRM2 and IMPDH2 inhibitors, previously shown to enhance the efficiency of TMZ. This novel and targetable interaction may present a promising clinical opportunity for GBM therapy.